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目的原代培养人主动脉瓣间质细胞并建立体外瓣膜细胞钙化模型,诱导人主动脉瓣间质细胞向成骨细胞分化,并观察其表型变化。方法采用胶原酶两次消化法原代培养人主动脉瓣间质细胞,取传代3～7代间质细胞,随机分为2组,实验组以钙化诱导培养基培养,对照组以标准培养基培养。1周后,行von Kossa染色观察钙化结节形成情况,分光光度计测定碱性磷酸酶活性,免疫荧光染色检测瓣膜间质细胞表型蛋白,real-time PCR及蛋白质印迹分析检测成骨相关因子的表达,评价模型建立情况。结果培养1周后实验组出现钙化结节,每孔钙化结节数量[(51.20±14.31)个]高于对照组[(3.60±1.82)个],差异有统计学意义(P<0.05),同时实验组碱性磷酸酶活性较对照组升高(约上升4倍,P<0.05),细胞收缩表型平滑肌肌动蛋白(α-SMA)增高。Real-time PCR及蛋白质印迹分析提示,实验组中成骨相关因子Runx2、osteocalcin及osteopontin在mRNA及蛋白水平均较对照组升高,磷酸化Smad1/5/8蛋白表达也同时升高,差异有统计学意义(P<0.05)。结论成功建立了人主动脉瓣间质细胞体外诱导钙化模型,诱导后间质细胞呈现相对激活状态,表型向收缩表型和成骨表型转化,为今后实验提供了可靠的细胞模型。
OBJECTIVE: To culture human aortic valve interstitial cells in primary culture and establish in vitro calcification model of valve cells to induce the differentiation of human aortic stromal cells into osteoblasts and observe the phenotypic changes. Methods The human aortic valve interstitial cells were primarily cultured by collagenase digestion twice. The passage 3 to 7 passage mesenchymal cells were randomly divided into 2 groups. The experimental group was cultured in calcification induction medium, and the control group was cultured in standard medium to cultivate. One week later, the formation of calcified nodules was observed by von Kossa staining. The alkaline phosphatase activity was measured by spectrophotometer. The expression of valvular mesenchymal cells phenotype was detected by immunofluorescence staining. Real-time PCR and Western blotting were used to detect the expression of osteogenesis-related factors Expression, evaluation of the establishment of the model. Results The number of calcified nodules in the experimental group after one week of culture was significantly higher than that in the control group [(51.20 ± 14.31)] [(3.60 ± 1.82)], with significant difference (P <0.05) At the same time, the activity of alkaline phosphatase in the experimental group was higher than that in the control group (about 4 times, P <0.05), and the contractile phenotype of smooth muscle actin (α-SMA) increased. Real-time PCR and Western blotting analysis indicated that the expressions of Runx2, osteocalcin and osteopontin in the experimental group were higher than those in the control group, and the expressions of phosphorylated Smad1 / 5/8 protein also increased at the same time Statistical significance (P <0.05). Conclusion The human aortic stromal cells in vitro induced calcification model was successfully established. After induction, the stromal cells showed a relative activation state, and the phenotype transformed into contractile phenotype and osteogenic phenotype, which provided a reliable cell model for future experiments.